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Long-term storage of feces at −80 °C versus −20 °C is negligible for 16S rRNA amplicon profiling of the equine bacterial microbiome

The development of next-generation sequencing technologies has spurred a surge of research on bacterial microbiome diversity and function. But despite the rapid growth of the field, many uncertainties remain regarding the impact of differing methodologies on downstream results. Sample storage temper...

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Autores principales: Gavriliuc, Stefan, Stothart, Mason R., Henry, Astrid, Poissant, Jocelyn
Formato: Online Artículo Texto
Lenguaje:English
Publicado: PeerJ Inc. 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7953882/
https://www.ncbi.nlm.nih.gov/pubmed/33854827
http://dx.doi.org/10.7717/peerj.10837
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author Gavriliuc, Stefan
Stothart, Mason R.
Henry, Astrid
Poissant, Jocelyn
author_facet Gavriliuc, Stefan
Stothart, Mason R.
Henry, Astrid
Poissant, Jocelyn
author_sort Gavriliuc, Stefan
collection PubMed
description The development of next-generation sequencing technologies has spurred a surge of research on bacterial microbiome diversity and function. But despite the rapid growth of the field, many uncertainties remain regarding the impact of differing methodologies on downstream results. Sample storage temperature is conventionally thought to be among the most important factors for ensuring reproducibility across marker gene studies, but to date much of the research on this topic has focused on short-term storage in the context of clinical applications. Consequently, it has remained unclear if storage at −80 °C, widely viewed as the gold standard for long-term archival of feces, is truly required for maintaining sample integrity in amplicon-based studies. A better understanding of the impacts of long-term storage conditions is important given the substantial cost and limited availability of ultra-low temperature freezers. To this end, we compared bacterial microbiome profiles inferred from 16S V3–V4 amplicon sequencing for paired fecal samples obtained from a feral horse population from Sable Island, Nova Scotia, Canada, stored at either −80 °C or −20 °C for 4 years. We found that storage temperature did not significantly affect alpha diversity measures, including amplicon sequence variant (ASV) richness and evenness, and abundance of rare sequence variants, nor presence/absence, relative abundances and phylogenetic diversity weighted measures of beta diversity. These results indicate that storage of equine feces at −20 °C for periods ranging from a few months to a few years is equivalent to storage at −80 °C for amplicon-based microbiome studies, adding to accumulating evidence indicating that standard domestic freezers are both economical and effective for microbiome research.
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spelling pubmed-79538822021-04-13 Long-term storage of feces at −80 °C versus −20 °C is negligible for 16S rRNA amplicon profiling of the equine bacterial microbiome Gavriliuc, Stefan Stothart, Mason R. Henry, Astrid Poissant, Jocelyn PeerJ Biodiversity The development of next-generation sequencing technologies has spurred a surge of research on bacterial microbiome diversity and function. But despite the rapid growth of the field, many uncertainties remain regarding the impact of differing methodologies on downstream results. Sample storage temperature is conventionally thought to be among the most important factors for ensuring reproducibility across marker gene studies, but to date much of the research on this topic has focused on short-term storage in the context of clinical applications. Consequently, it has remained unclear if storage at −80 °C, widely viewed as the gold standard for long-term archival of feces, is truly required for maintaining sample integrity in amplicon-based studies. A better understanding of the impacts of long-term storage conditions is important given the substantial cost and limited availability of ultra-low temperature freezers. To this end, we compared bacterial microbiome profiles inferred from 16S V3–V4 amplicon sequencing for paired fecal samples obtained from a feral horse population from Sable Island, Nova Scotia, Canada, stored at either −80 °C or −20 °C for 4 years. We found that storage temperature did not significantly affect alpha diversity measures, including amplicon sequence variant (ASV) richness and evenness, and abundance of rare sequence variants, nor presence/absence, relative abundances and phylogenetic diversity weighted measures of beta diversity. These results indicate that storage of equine feces at −20 °C for periods ranging from a few months to a few years is equivalent to storage at −80 °C for amplicon-based microbiome studies, adding to accumulating evidence indicating that standard domestic freezers are both economical and effective for microbiome research. PeerJ Inc. 2021-03-09 /pmc/articles/PMC7953882/ /pubmed/33854827 http://dx.doi.org/10.7717/peerj.10837 Text en © 2021 Gavriliuc et al. https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ) and either DOI or URL of the article must be cited.
spellingShingle Biodiversity
Gavriliuc, Stefan
Stothart, Mason R.
Henry, Astrid
Poissant, Jocelyn
Long-term storage of feces at −80 °C versus −20 °C is negligible for 16S rRNA amplicon profiling of the equine bacterial microbiome
title Long-term storage of feces at −80 °C versus −20 °C is negligible for 16S rRNA amplicon profiling of the equine bacterial microbiome
title_full Long-term storage of feces at −80 °C versus −20 °C is negligible for 16S rRNA amplicon profiling of the equine bacterial microbiome
title_fullStr Long-term storage of feces at −80 °C versus −20 °C is negligible for 16S rRNA amplicon profiling of the equine bacterial microbiome
title_full_unstemmed Long-term storage of feces at −80 °C versus −20 °C is negligible for 16S rRNA amplicon profiling of the equine bacterial microbiome
title_short Long-term storage of feces at −80 °C versus −20 °C is negligible for 16S rRNA amplicon profiling of the equine bacterial microbiome
title_sort long-term storage of feces at −80 °c versus −20 °c is negligible for 16s rrna amplicon profiling of the equine bacterial microbiome
topic Biodiversity
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7953882/
https://www.ncbi.nlm.nih.gov/pubmed/33854827
http://dx.doi.org/10.7717/peerj.10837
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